The Renal Outer Medullary K+ channel, ROMK (Kir1.1, KCNJ1), plays key physiological roles in maintenance of systemic electrolyte and water homeostasis by the kidney. ROMK function is critical for salt re-absorption and is thought to form the dominant pathway for K+ secretion in the kidney nephron. The importance of ROMK is underscored by the identification of heritable loss-of-function ROMK mutations in patients with type Bartter's syndrome, a severe kidney tubule disorder characterized by salt and water wasting and acid-base disturbances. Given the pivotal role of ROMK in kidney function and disease, the identification of novel ROMK modulators is highly desirable and could provide valuable tools for basic research and the treatment of numerous kidney-related disorders. However, currently available assays of ROMK function are slow, labor intensive and therefore not amenable to high-throughput screening (HTS) of chemical libraries. Furthermore, a significant barrier to the development of high-throughput (HT)-compatible assays of ROMK function has been the inability to express sufficient ROMK in the plasma membrane of transfected cells. To circumvent this limitation, we have engineered a single point mutation (S44D) into the cytoplasmic N- terminus of ROMK that allows expression of robust K+ currents in transfected HEK-293 cells. Importantly, we further show that ROMK-S44D activity can be monitored in 384-well format using a novel fluorescence- based assay of channel function. In this grant application, we propose to 1) develop a fluorescence-based assay of ROMK activity using ROMK-S44D as a surrogate channel. A detailed characterization of ROMK- S44D function will be performed to test its utility as a surrogate for the wild type channel. We will also test the effects of the known ROMK inhibitor tertiapin-Q on fluorescence signals generated by the movement of thallium (Tl+) through ROMK-S44D expressed in HEK-293 cells loaded with the Tl+-sensitive dye BTC. 2) Validate the fluorescence assay for use in HTS. This will be done by performing a small scale, 10,000 compound validation screen to identify lead compounds for secondary HT screens. Lay Summary: The development of high throughput assays for ROMK function holds significant potential for the identification of novel compounds that could be used in basic science discovery as well as clinical medicine for the management of hypertension, edema and other kidney disease-related disorders. [unreadable] [unreadable] [unreadable]